Antibiotic U-53,946 and process for preparing the same

ABSTRACT

New antiobiotic U-53,946 produced by the controlled fermentation of the new microorganism Paecilomyces abruptus sp. nov., NRRL 11110. This antibiotic is active against Gram-positive bacteria, for example, Sarcina lutea, Staphylococcus aureus, Bacillus subtilis, Mycobacterium avium, and Streptococcus pyrogenes, and the yeast Saccharomyces pastorianus. Accordingly, they can be used in various environments to eradicate or control these microorganisms.

The invention described herein was made in the course of, or underContract NO1-CM-43753 with the National Cancer Institute, NationalInstitutes of Health, Bethesda, MD. 20014.

BRIEF SUMMARY OF THE INVENTION

The novel antibiotic of the invention, U-53,946 is obtained by culturingPaecilomyces abruptus sp. nov. NRRL 11110, in an aqueous nutrient mediumunder aerobic conditions. Antibiotic U-53,946 has the property ofadversely affecting the growth of Gram-positive bacteria, for example,S. aureus, S. lutea, B. subtilis, M. avium, and S. pyogenes.Accordingly, U-53,946 can be used alone or in combination with otherantibiotic agents to prevent the growth of or reduce the number ofbacteria, as disclosed above, in various environments.

DETAILED DESCRIPTION OF THE INVENTION Chemical and Physical Propertiesof U-53,946

Molecular Formula: C₆₁ H₁₀₇ N₁₁ O₁₄

Elemental Analysis: Calcd. C, 60.12; H, 8.85; N, 12.65; O, 18.38. Found:C, 60.13, 59.73; H, 9.21, 8.92; N, 12.16, 11.97; O, 18.44.

Molecular Weight: 1218 ± 1 (Determined by mass spectrometry)

Melting Point: 200°-205° C.

Specific Rotation: [α]_(D) ²⁵ = -26° (c, 0.9175 EtOH)

Solubilities: Antibiotic U-53,946 is soluble in water and loweralcohols, for example, methanol and butanol; ketones, for example,methyl ethyl ketone, halogenated solvents, ethyl acetate, or otherester-type solvents; it is relatively insoluble in aliphatichydrocarbons.

Antibiotic U-53,946 is a neutral compound.

Ultraviolet Absorption Spectra: In ethanol-max 245 nm ε 1510 (see FIG. 2of the drawings). R_(f) : Antibiotic U-53,946 has an R_(f) of 0.34(CHCl₃ --CH₃ OH--NH₄ OH; 85:14:1 v/v).

Infrared Absorption Spectrum: U-53,946 has a characteristic infraredabsorption spectrum in a mineral oil mull as shown in FIG. 1 of thedrawings. Peaks are observed at the following wave lengths expressed inreciprocal centimeters:

    ______________________________________                                        Band Frequency                                                                (Wave Numbers)                                                                              Intensity                                                       ______________________________________                                        3450          M, sh       (sh=shoulder)                                       3320          S           (S=strong)                                          3040          W           (M=medium)                                          2950          S           (W=weak)                                            2920          S                                                               2850          S                                                               2730          W                                                               1710          M, sh                                                           1592          M                                                               1537          S                                                               1460          S                                                               1445          M, sh                                                           1385          M                                                               1377          M                                                               1365          M                                                               1307          M                                                               1292          M                                                               1223          M                                                               1190          W                                                               1173          W                                                               1150          W                                                               1125          W                                                               1070          W                                                               1037          W                                                               1015          W                                                               982           W                                                               952           W                                                               923           W                                                               908           W                                                               837           W                                                               720           W                                                               708           W                                                               ______________________________________                                    

    ______________________________________                                        Antimicrobial Activity of U-53,946                                                                         Zone of                                          Microorganism      Medium*   Inhibition (mm)                                  ______________________________________                                        Proteus vulgaris   1         0                                                Salmonella gallinarum                                                                            1         0                                                Pseudomonas aeruginosa                                                                           1         0                                                Salmonella schottmuelleri                                                                        1         0                                                Klebsiella pneumoniae                                                                            2         0                                                Sarcina lutea      3         31                                               Staphylococcus aureus                                                                            1         25                                               Bacillus subtilis  2         25                                               Mycobacterium avium                                                                              4         30                                               Streptococcus pyogenes                                                                           4         33                                               Saccharomyces pastorianus                                                                        5         17                                               Penicillium oxalicum                                                                             6         0                                                ______________________________________                                         *Medium 1(Nutrient Agar), 2(Streptomycin Assay Agar), 3(Seed Agar),           4(Brain Heart Infusion Agar), and 6(Malt Extract Agar) can be obtained        from Difco Company, Detroit, Michigan. Medium 5(Gray's Agar) has the          following ingredients:                                                   

    Gm/liter H.sub.2 O                                                            ______________________________________                                        Glucose             30                                                        Yeast Extract        7                                                        KH.sub.2 PO.sub.4    5                                                        Agar                15                                                        ______________________________________                                    

The above antimicrobial spectrum was obtained by a standard disc platemethod using 13 mm paper discs. The microorganisms were cultivated inmedia as shown beneath the results, supra. The preparation of U-53,946was tested at a concentration of 1 mg/ml.

A solution of this preparation in water (0.08 mcg/ml) inhibited L 1210cell growth in vitro by 90%.

THE MICROORGANISM

The microorganism used for the production of U-53,946 is Paecilomycesabruptus sp. nov., NRRL 11110. A subculture of this microorganism can beobtained from the permanent collection of the Northern Regional ResearchLaboratory, U.S. Department of Agriculture, Peoria, Ill. U.S.A. Itshould be understood that the availability of the culture does notconstitute a license to practice the subject invention in derogation ofpatent rights granted with the subject instrument by governmentalaction.

The microorganism of this invention was studied and characterized byAlma Dietz and Grace P. Li of The Upjohn Research Laboratories.

A soil isolate (designated CC-1014) was considered to belong to thePenicillium lilacinum Series of Raper and Thom's [Raper, K.B. and C.Thom. 1949. A Manual of the Penicillia. 875 pp. The Williams and WilkinsCo., Baltimore] section Assymmetrica, sub-section Divaricata or to thegenus Paecilomyces. P. lilacinum and Paecilomyces both lack the greencolor associated with most penicillia but produce branched conidialstructures like those found in Penicillium species. Therefore, the newisolate was compared with the P. lilacinum strain in our collection. Thecultures showed aerial growth color similarities on most media butdiffered in reverse color and microscopic properties as may be noted inthe tables that follow. Significant microscopic characteristics of thenew culture are the appearance of a typical penicillus, a short (abrupt)conidiophore from which arise a cluster of 3-5 sterigmata of equallength. The sterigmata appear as tapered tubes which give rise to longchains of smooth surfaced elliptical spores. Another characteristic ofthe new isolate is the production of antibiotic U-53,946 (a cytotoxicagent, CC-1014).

DESCRIPTION Paecilomyces abruptus Dietz and Li, sp. nov.

Color Characteristics. The appearance of the cultures on Ektachrome isgiven in Table 1. Reference Color Characteristics of the cultures onagar are given in Table 2.

Temperature Studies. Both cultures grew well at 18°-32° C.

Paecilomyces abuptus grew well at 37° C. The properties cited for thenew species are confirmatory for the Penicillium lilacinum series or thePaecilomyces group of Raper and Thom, supra. The P. lilacinum series isnow assigned to the genus Paecilomyces [Index of Fungi. 1975. A List ofNames of New Genera, Species and Varieties of Fungi and Lichens. NewCombinations and New Names, Compiled from World Literature. 4: 283.Commonwealth Mycological Institute. Kew. Surrey.]. Macroscopic andmicroscopic properties noted for the new soil isolate enable it to bedistinguished from the species assigned to this genus for whichdescriptions are available. Therefore, we propose that the new strain bedesignated Paecilomyces abruptus Dietz and Li, sp. nov. The speciesdesignation is based on the distinctive short (abrupt) conidiophores ofthis isolate.

Paecilomyces lilacinus grew poorly at 37° C. Good growth occurred whenthe plates were reincubated at 24° C. The cultures did not grow at 45°or 55° C. These temperatures were fungicidal.

Microscopic Characteristics. The cultures are compared in Table III.

METHODS

Macroscopic and microscopic growth characteristics were determined onmedia cited in Cooke [Cooke, W.B. 1963. A Laboratory Guide to Fungi inPolluted Waters, Sewage, and Sewage Treatment Systems. TheirIdentification and Culture. Public Health Service Publication No.999-WP-1. U.S. Dept. of Health, Education, and Welfare. Public HealthService. Division of Water Supply and Pollution Control. Cincinnati 26,Ohio], Raper and Thom [Raper, K.B. and C. Thom. 1949. A Manual of thePenicillia. 875 pp. The Williams and Wilkins Co., Baltimore], and Smith[Smith, G. 1946. 3rd ed. An Introduction to Industrial Mycology. EdwardArnold and Co., Ltd., London. (Reprinted 1947. Jarrold and Sons, Ltd.,Norwich)].

The soil isolate and Paecilomyces lilacinus (formerly Penicilliumlilacinum) BB-156 (UC 4371), the culture in our collection to which thenew isolate appeared most similar, were seeded from soil stocks toGray's broth in shake flasks. The seeded flasks were incubated for 48hours on a reciprocal shaker at 28° C. Growth in the shake flasks wasblended for one minute at low speed in a Waring Blender. The blendedinoculum was seeded on slants and plates. Agar slant media used wereNeopeptone-Dextrose, Czapek's Sucrose, Leonian's, Water, Potato-Dextroseand Gray's. Agar plate media were: Czapek's Sucrose, Potato-Dextrose,Malt Extract for single plates and the same media plus Wort Agar forfour-sector plates. Slants and single plates were incubated at 28° C.Four-sector plates were incubated at 18°, 24°, 28°, 32°, 37°, 45°, and55° C.

The color pattern of the growth on the slant media was photographed onEktachrome after seven days incubation at 28° C. Color determination ofagar plate growth was made using the ISCC-NBS Centroid Color Charts(Supplement to NBS Circular 553 [Kelley, K. L. and D. B. Judd. 1955. TheISCC-NBS Method of Designating Colors and a Dictionary of Color Names.National Bureau of Standards Circular 553. Superintendant of Documents,U.S. Government Printing Office, Washington, D.C.]).

Microscopic characteristics were determined by dissecting microscopeexamination and by scanning electron microscope examination using themethods of Dietz and Mathews [Dietz, A. and J. Mathews. 1969. Scanningelectron microscopy of selected members of the Streptomyceshygroscopicus group. Appl. Microbiol. 18: 694-696].

NOTE: The designation "UC", which appears in this specification, priorto a number refers to The Upjohn Company Culture Collection and is aregistered trademark.

                  Table I                                                         ______________________________________                                        Appearance of Cultures on Ektachrome                                                   De-                                                                           ter-                                                                          mi-     Paecilomyces  Paecilomyces                                   Agar     na-     abruptus      lilacinus                                      Medium   tion    NRRL 11110    BB-156, UC 4371                                ______________________________________                                        Neopeptone-                                                                            S       Lavender-gray Lavender-gray                                  Dextrose                                                                               R       Cream         Yellow-tan                                     Czapek's S       Lavender-gray-pink                                                                          Lavender-gray-pink                             Sucrose                                                                                R       Cream         Red-tan                                        Leonian's                                                                              S       Pink          Pink                                                    R       Pale pink     Pink-tan                                       Water    S       Trace lavender-pink                                                                         Trace lavender-pink                                     R       Gray-pink     Gray-pink                                      Potato-  S       Pink          Lavender-pink                                  Dextrose                                                                               R       Cream-tan     Red-tan                                        Gray's   S       Lavender-pink Lavender-pink                                           R       Yellow-tan    Red-tan                                        ______________________________________                                         S = Surface color                                                             R = Reverse color                                                        

                  Table II                                                        ______________________________________                                        Reference Color Characteristics*                                              De-       Paecilomyces   Paecilomyces                                         ter-      abruptus       lilacinus                                            mi-       NRRL 11110     BB-156 UC 4371                                       Agar   na-    Chip   Color     Chip Color                                     Medium tion   No.    Description                                                                             No.  Description                               ______________________________________                                        Czapek's                                                                             S      32     Grayish yel-                                                                            9    Pinkish white                             Sucrose              lowish pink                                                     R       9     Pinkish white                                                                           31   Pale yellowish                                                                pink                                      Gray's S      32     Grayish yel-                                                                            9    Pinkish white                                                  lowish pink                                                     R      89     Pale yellow                                                                             79   Light grayish                                                                 yellowish brown                                                               to                                                                       46   Grayish reddish                                                               brown                                     Malt   S      32     Grayish yel-                                                                            9    Pinkish white                             Extract              lowish pink                                                     R      33     Brownish pink                                                                           73   Pale orange                                                    (center)       yellow                                                  92     Yellowish                                                                     white (edge)                                             Potato-                                                                              S      32     Grayish yel-                                                                            9    Pinkish white                             Dextrose             lowish pink                                                     R      79     Light grayish                                                                           46   Grayish reddish                                                yellowish      brown (trace)                                                  brown          with                                                                     33   Brownish pink                                                                 to                                                                       20   Dark grayish red                                                              to                                                                       31   Pale yellowish                                                                pink                                      Wort   S      32     Grayish yel-                                                                            9    Pinkish white                                                  lowish pink                                                     R      58     Moderate  70   Light orange                                                   brown to       yellow                                                  76     Light yellow-                                                                 ish brown                                                ______________________________________                                         *Kelley, K.L. and D.B. Judd., supra.                                          S = Surface color                                                             R = Reverse color                                                        

                  Table III                                                       ______________________________________                                        Microscopic Characteristics                                                              Paecilomyces                                                                              Paecilomyces                                                      abruptus    lilacinus                                                         NRRL 11110  BB-156, UC 4371                                        ______________________________________                                        Dissecting                                                                    Microscope                                                                    Colony type  Velvety to lanose.                                                                          Velvety.                                           Colony growth                                                                              Rapid-difficult to                                                                          Rapid-difficult to                                              determine edge.                                                                             determine edge.                                                 Central area raised                                                                         --                                                              and radically                                                                 furrowed.                                                        Zonation     --            --                                                 Exudate      Good to heavy on                                                                            Poor on Czapek's                                                Czapek's sucrose                                                                            sucrose and malt-                                               and malt-extract                                                                            extract agars.                                                  agars. Poor on                                                                              Good on potato-                                                 potato-dextrose                                                                             dextrose agar.                                                  agar.                                                            Scanning Electron                                                             Microscope                                                                    Conidiophores                                                                              7.5 × 2.5 μm.                                                                      7.5 × 2 μm.                                            Smooth.       Smooth.                                                         Arise from    Arise from substrate                                            substrate and and aerial                                                      aerial hyphae.                                                                              hyphae.                                                         May be terminal.                                                                            May be terminal.                                   Metulae      Uncommon.     Uncommon.                                          Sterigmata   3-5.          Usually 2.                                                      Tapered.      Tapered.                                                        7.0 × 2.5 μm to                                                                    7.0 × 2.0 μm                                           7.0 × 1.0 μm.                                                                      to 7.0 × 0.7 μm.                          Conidial chains                                                                            Parallel.     Not usually parallel.                              Conidia      Smooth.       Smooth.                                                         3.0 × 1.75 μm.                                                                     2 × 1.25 μm.                                           Ridged appearance                                                                           Ridged appearance                                               from 2-sided  from 2-sided                                                    dimpling.     dimpling.                                          Sclerotia    Not detected. Not detected.                                      Perithecia   Not detected. Not detected.                                      Ascospores   Not detected. Not detected.                                      ______________________________________                                    

The new compound of the invention is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood, also, that for the preparation oflimited amounts surface cultures and bottles can be employed. Theorganism is grown in a nutrient medium containing a carbon source, forexample, an assimilable carbohydrate, and a nitrogen source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include cornsteep liquor, yeast, autolyzedbrewer's yeast with milk solids, soybean meal, cottonseed meal,cornmeal, milk solids, pancreatic digest of casein, fish meal,distillers' solids, animal peptone liquors, meat and bone scraps, andthe like. Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

Production of the compound of the invention can be effected at anytemperature conducive to satisfactory growth of the microorganism, forexample, between about 18° and 40° C., and preferably between about 20°and 28° C. Ordinarily, optimum production of the compound is obtained inabout 3 to 15 days. The medium normally remains neutral during thefermentation. The final pH is dependent, in part, on the bufferspresent, if any and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the new compound and the attendant inefficient utilizationof the equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating this broth culturewith an aliquot from a soil, liquid N₂ agar plug, or a slant culture.When a young, active vegetative inoculum has thus been secured, it istransferred aseptically to large vessels or tanks. The medium in whichthe vegetative inoculum is produced can be the same as, or differentfrom, that utilized for the production of the new compound, so long as agood growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation andpurification of the compound of the subject invention, for example,solvent extraction, partition chromatography, silica gel chromatography,liquid-liquid distribution in a Craig apparatus, adsorption on resins,and crystallization from solvents.

In a preferred recovery process the compound of the subject invention isrecovered from the culture medium by separation of the mycelia andundissolved solids by conventional means, such as by filtration orcentrifugation. The antibiotic is recovered from the filtered orcentrifuged broth by extraction with a solvent for U-53,946, forexample, ethyl acetate (preferred), n-butanol, methyl ethyl ketone,chloroform, and the like. The extraction is carried on after thefiltered beer is adjusted to a pH of about 7 to about 10 with a base,for example, sodium hydroxide.

Essentially pure antibiotic U-53,946 can be obtained from U-53,946preparations, obtained as disclosed above, by chromatographicprocedures. In a preferred process, preparations of antibiotic U-53,946are subjected to chromatographic procedures using silica gel and thesolvent system chloroform-methanol-ammonium hydroxide (86:13:1 v/v).Active fractions as determined by assay against S. aureus are combinedand evaporated to dryness to give a solid preparation of U-53,946 havingan R_(f) of 0.30.

Antibiotic U-53,946 is active against S. aureus and can be used todisinfect washed and stacked food utensils contaminated with thisbacteria; they can also be used as disinfectants on various dental andmedical equipment contaminated with S. aureus.

Further, U-53,946 can be used for treating breeding places of silkworms,to prevent or minimize infections which are well known to be caused byBacillus subtilis.

Still further, U-53,946 can be used to control Mycobacterium avium whichis a known producer of generalized tuberculosis in birds and rabbits.

It is to be understood that the microbiological process disclosedherein, though described in detail with reference to Paecilomycesabruptus sp. nov., NRRL 11110, is not limited to this particularmicroorganism deposit. It is intended that any microorganism meeting thecultural characteristics disclosed herein, or substantial equivalencethereof, wherever deposited in the world, is a part of the subjectmicrobiological process. Further, it is intended that this inventioninclude strains or mutants of the said microorganism which can beproduced by procedures well known in the art, for example, by subjectingthe novel microorganism to x-ray or ultraviolet radiation, nitrogenmustard, phage exposure, and the like.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Part A. Fermentation

An agar slant of Paecilomyces abruptus sp. nov., NRRL 11110, is used toinoculate a series of 500-ml Erlenmyer flasks, each containing 100 ml ofsterile seed medium consisting of the following ingredients:

    ______________________________________                                        Glucose monohydrate    25 gm/l                                                Pharmamedia*           25 gm/l                                                Tap water q.s.         1 liter                                                ______________________________________                                         *Pharmamedia is an industrial grade of cottonseed flour produced by           Traders Oil Mill Company, Fort Worth, Texas.                             

The seed medium presterilization pH is 7.2. The seed inoculum is grownfor two days at 28° C. on a Gump rotary shaker operating at 250 r.p.m.and having a 21/2 inch stroke.

After 2 days incubation, the seed medium is used to inoculate (theinoculation rate is 5 ml of seed inoculum per 100 ml of fermentationmedium) each of 500 ml non-stippled flasks each containing 100 ml ofsterile fermentation medium consisting of the following ingredients:

    ______________________________________                                        Glucose monohydrate    10 gm/l                                                Dextrin                10 gm/l                                                Soytone*               5 gm/l                                                 Distillers soluble**   5 gm/l                                                 Malt extract           5 gm/l                                                 Tap water q.s.         1 liter                                                pH - 7.2 (presterilization)                                                   ______________________________________                                         *Soytone (Produced by Difco Laboratories, Detroit, Mich.).                    **Distillers soluble (Brown-Forman Distillers Corp., P. O. Box 1080,          Louisville, KY 40201.                                                    

The fermentation flask are incubated at a temperature of 28° C. on aGump rotary shaker operating at 250 r.p.m. and having a 21/2 inchstroke.

Part B. Recovery

Antibiotic U-53,946 in beers in detected and assayed by the use of thinlayer chromatography (tlc) and antibacterial assays. Thin layerchromatograms are run on silica gel plates usingchloroform-methanol-ammonium hydroxide (86:13:1 v/v) as the solventsystem. Bioactivity is detected by bioautography using standard S.aureus seeded agar trays.

Whole fermentation beer (ca. 1740 ml), obtained as described above, isfiltered with the aid of diatomaceous earth as a filter aid. The filtercake is washed with 400 ml of water. The cake is discarded. The filtrate(1700 ml, pH 8.0) is adjusted to pH 10.0 with a 1.0 N sodium hydroxidesolution. The aqueous solution is extracted with four 800-ml portions ofethyl acetate. The combined ethyl acetate extracts are evaporated underreduced pressure to a solid residue preparation of U-53,946; yield, 334mg. A solution of this preparation in water (0.08 mcg/ml) inhibited L1210 cell growth in vitro by 90%.

Part C. Purification

Nine hundred and thirty-nine mg of a solid preparation of U-53,946,obtained as disclosed above, is chromatographed on 100 g of silica gelusing the solvent system chloroform-methanol-ammonium hydroxide(86:13:1) and collecting two hundred and fifty-five 5-ml fractions.Fractions 65-118 are combined and evaporated under reduced pressure togive 248 mg of product. This residue contains two materials as indicatedby tlc on silica plates in chloroform-methanol-ammonium hydroxide(85:14:1). A second pool consisting of fractions 119-155 is evaporatedunder reduced pressure to give 300 mg of U-53,946 homogeneous by tlc asabove, R_(f) 0.30. The product obtained from pool I isrechromatographed, as discussed above. Those fractions containing pureU-53,946 as shown by tlc are combined and evaporated to dryness underreduced pressure; yield, 164 mg.

We claim:
 1. Antibiotic U-53,946, which is active against Gram-positivebacteria, and which in its essentially pure form:(a) has the molecularformula C₆₁ H₁₀₇ N₁₁ O₁₄ ; (b) has the following elemental analysis: C,60.13, 59.73; H, 9.21, 8.92; N, 12.16, 11.97; O, 18.44; (c) has aspecific rotation of [α]_(D) ²⁵ = -26° (c, 0.9175 EtOH); (d) is solublein lower alcohols, for example, methanol, ethanol and butanol; ketones,for example, methyl ethyl ketone, halogenated solvents, ethyl acetate,or other ester-type solvents; and is relatively insoluble in aliphatichydrocarbons; (e) has a characteristic infrared adsorption spectrum whendissolved in a mineral oil mull as shown in FIG. 1 of the drawings; and,(f) has a characteristic UV spectrum as shown in FIG. 2 of the drawings.2. A process for preparing antibiotic U-53,946 which comprisescultivating Paecilomyes abruptus sp. nov., having the identifyingcharacteristics of NRRL 11110, in an aqueous nutrient medium underaerobic conditions until substantial antibiotic activity is imparted tosaid medium, and recovering antibiotic U-53,946.
 3. A process, accordingto claim 2, wherein said aqueous nutrient medium contains a source ofassimilable carbohydrate and assimilable nitrogen.
 4. A process forrecovering antibiotic U-53,946, according to claim 2, whichcomprises:(a) filtering the fermentation beer to obtain a clear beer;(b) adjusting the pH of the clear beer to about 7 to about 10; (c)extracting said clear beer with a solvent for U-53,946 to obtain a solidpreparation of antibiotic U-53,946; and, (d) chromatographing said solidpreparation of antibiotic U-53,946 on silica gel usingchloroform-methanol-ammonium hydroxide (86:13:1 v/v) as the solvent toobtain an essentially pure preparation of antibiotic U-53,946.
 5. Aprocess, according to claim 5, wherein the extraction solvent is ethylacetate.